Ligand-Directed Modification of Active Matrix Metalloproteases: Activity-based Probes with no Photolabile Group.

Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S3 ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

Compression of Large Sets of Sequence Data Reveals Fine Diversification of Functional Profiles in Multigene Families of Proteins: A Study for Peptidyl-Prolyl cis/trans Isomerases (PPIase).

In this technical note, we describe analyses of more than 15,000 sequences of FK506-binding proteins (FKBP) and cyclophilins, also known as peptidyl-prolyl cis/trans isomerases (PPIases). We have developed a novel way of displaying relative changes of amino acid (AA)-residues at a given sequence position by using heat-maps. This type of representation allows simultaneous estimation of conservation level in a given sequence position in the entire group of functionally-related paralogues (multigene family of proteins). We have also proposed that at least two FKBPs, namely FKBP36, encoded by the Fkbp6 gene and FKBP51, encoded by the Fkbp5 gene, can form dimers bound via a disulfide bridge in the nucleus. This type of dimer may have some crucial function in the regulation of some nuclear complexes at different stages of the cell cycle.

Introduction to Peptidyl-Prolyl cis/trans Isomerase (PPIase) Series.

About 30 years after the discovery of peptidyl-prolyl cis/trans isomerases (PPIases), research on this group of proteins has become somewhat calmer than it used to be, but it still generates lots of interest [...].

Peptidylprolyl Isomerases as In Vivo Carriers for Drugs That Target Various Intracellular Entities.

Analyses of sequences and structures of the cyclosporine A (CsA)-binding proteins (cyclophilins) and the immunosuppressive macrolide FK506-binding proteins (FKBPs) have revealed that they exhibit peculiar spatial distributions of charges, their overall hydrophobicity indexes vary within a considerable level whereas their points isoelectric (pIs) are contained from 4 to 11. These two families of peptidylprolyl cis/trans isomerases (PPIases) have several distinct functional attributes such as: (1) high affinity binding to some pharmacologically-useful hydrophobic macrocyclic drugs; (2) diversified binding epitopes to proteins that may induce transient manifolds with altered flexibility and functional fitness; and (3) electrostatic interactions between positively charged segments of PPIases and negatively charged intracellular entities that support their spatial integration. These three attributes enhance binding of PPIase/pharmacophore complexes to diverse intracellular entities, some of which perturb signalization pathways causing immunosuppression and other system-altering phenomena in humans.

Multidimensional Drift of Sequence Attributes and Functional Profiles in the Superfamily of the Three-Finger Proteins and Their Structural Homologues.

Functional diversity of the three-finger-protein domain (TFPD) had been acquired via hypervariability of some sequence positions and extensive insertion/deletion of short AA-segments that caused multidimensional drift of several sequence attributes such as the overall (HI) and local hydrophobicity levels, the isoelectric point (pI), distribution of charges, and local polarizability potentials. In consequence, the pIs of various TFPDs vary from 3 to 10, and the HIs span from highly hydrophilic to extreme hydrophobic levels. Our analyses of diverse genomic databases suggest that a primordial TFP-like fold could have been adapted to extracellular N-terminal domain (ECD) of the TGFß series of receptors that are crucial for morphogenesis of multicellular organisms with elaborated body plans. It seems plausible that from the exons coding for the primordial ECD had radiated some ORFs that gave rise to small monodomain and multidomain TFPs that may be either membrane-bound or soluble entities and whose repertoire expanded in the genomes of vertebrates. We show that the above-mentioned attributes have been restrained within narrow ranges in several functionally related groups of TFPs that are expressed in miscellaneous organisms.

Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities.

In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

Diversified targets of FKBP25 and its complex with rapamycin.

FKBP25 is a member of the super-family of peptidylprolyl cis/trans isomerases, which is a high affinity binder for the immunosuppressive antibiotic rapamycin (Rpm). FKBP25 isolated from natural sources, its recombinant murine homologue (mFKBP25) and their complexes with rapamycin bind to diverse DNAs, RNAs and heparin affinity beads. The recombinant mFKBP25/rapamycin complex binds to several proteins including the calcineurin-A/calcineurin-B/calmodulin complex and to elongation factor 1ß. We solved the X-ray structure of the C-terminal domain of mFKBP25 bound to rapamycin that has a higher resolution than of its human counterpart, and which clearly illustrates that the positively charged 40s loop is an epitope of the FK506-like binding domain (FKBD) for interactions with various biopolymers.

Functional diversity and pharmacological profiles of the FKBPs and their complexes with small natural ligands.

From 5 to 12 FK506-binding proteins (FKBPs) are encoded in the genomes of disparate marine organisms, which appeared at the dawn of evolutionary events giving rise to primordial multicellular organisms with elaborated internal body plan. Fifteen FKBPs, several FKBP-like proteins and some splicing variants of them are expressed in humans. Human FKBP12 and some of its paralogues bind to different macrocyclic antibiotics such as FK506 or rapamycin and their derivatives. FKBP12/(macrocyclic antibiotic) complexes induce diverse pharmacological activities such as immunosuppression in humans, anticancerous actions and as sustainers of quiescence in certain organisms. Since the FKBPs bind to various assemblies of proteins and other intracellular components, their complexes with the immunosuppressive drugs may differentially perturb miscellaneous cellular functions. Sequence-structure relationships and pharmacological profiles of diverse FKBPs and their involvement in crucial intracellular signalization pathways and modulation of cryptic intercellular communication networks were discussed.

Molecular aspects of cyclophilins mediating therapeutic actions of their ligands.

Cyclosporine A (CsA) is an immunosuppressive cyclic peptide that binds with a high affinity to 18 kDa human cyclophilin-A (hCyPA). CsA and its several natural derivatives have some pharmacological potential in treatment of diverse immune disorders. More than 20 paralogues of CyPA are expressed in the human body while expression levels and functions of numerous ORFs encoding cyclophilin-like sequences remain unknown. Certain derivatives of CsA devoid of immunosuppressive activity may have some potential in treatments of Alzheimer diseases, Hepatitis C and HIV infections, amyotrophic lateral sclerosis, congenital muscular dystrophy, asthma and various parasitic infections. Here, we discuss structural and functional aspects of the human cyclophilins and their interaction with various intra-cellular targets that can be under the control of CsA or its complexes with diverse cyclophilins that are selectively expressed in different cellular compartments. Some molecular aspects of the cyclophilins expressed in parasites invading humans and causing diseases were also analyzed.

On transversal hydrophobicity of some proteins and their modules.

Hydrophobicity of proteins encoded in the genomes of diverse organisms was quantified using two novel concepts: (A) amino acid (AA) bulkiness-dependent hydrophobicity profiles and (B) spatial context of hydrophobicity distribution in AA triads. Both concepts were introduced into an algorithm that was used for extracting protein clusters from diverse genomic databases whose sequence attributes were similar to those in the multiple sequence alignment (MSA) of a given family of proteins. The sequences of the G protein-coupled receptors (GPCRs) encoded in different genomes were used as templates for testing the above concepts. The following sequence attributes were used for protein clustering: (A) sequence similarity scores (IDs); (B) amino acid composition (AAC); (C) hydrophobicity; (D) AA-bulkiness; and (E) alpha-helical propensity potentials. Diverse GPCRs display variable distributions of AA bulkiness-dependent buildups and declines in the hydrophobicity profiles that may be related to their function-dependent way of packing and allostery in the membrane. It is shown that intramolecular transversal nonbonded interactions between the TM segments in diverse GPCRs involve about 50% of hydrophobic atoms. Similar interaction networks exist between alpha-helices of tetratricopeptide (TPR) motifs-containing immunophilins and other proteins containing alpha-helical bundles.

Conserved structural determinants in three-fingered protein domains.

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.

Functional drift of sequence attributes in the FK506-binding proteins (FKBPs).

Diverse members of the FK506-binding proteins (FKBPs) group and their complexes with different macrocyclic ligands of fungal origins such as FK506, rapamycin, ascomycin, and their immunosuppressive and nonimmunosuppressive derivatives display a variety of cellular and biological activities. The functional relatedness of the FKBPs was estimated from the following attributes of their aligned sequences: 1 degrees conservation of the consensus sequence; 2 degrees sequence similarity; 3 degrees pI; 4 degrees hydrophobicity; 5 degrees amino acid hydrophobicity and bulkiness profiles. Analyses of the multiple sequence alignments and intramolecular interaction networks calculated from a series of structures of the FKBPs revealed some variations in the interaction clusters formed by the AA residues that are crucial for sustaining peptidylprolyl cis/trans isomerases (PPIases) activity and binding capacity of the FKBPs. Fine diversification of the sequences of the multiple paralogues and orthologues of the FKBPs encoded in different genomes alter the intramolecular interaction patterns of their structures and allowed them to gain some selectivity in binding to diverse targets (functional drift).

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action.

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.

Molecular cloning and expression pattern of the Fkbp25 gene during cerebral cortical neurogenesis.

Neocortical neurons are generated predominantly from the cells that proliferate in the ventricular zone of the telencephalon. In order to understand the nature of these expanding cortical neuronal progenitor cells, we selected by differential display some transcripts that were enriched in the telencephalon as compared to the more caudal regions (diencephalon/mesencephalon). This systematic screening revealed one of the differentially expressed transcripts, namely the Fkbp25 mRNA that encodes a member of the FK506 binding proteins (FKBPs). Northern blot analysis showed that the expression of the single 1.4kb Fkbp25 transcript reached a maximum level on embryonic day 11.5 at the start of cortical neurogenesis in the mouse and was followed by a weak basal expression in the adult brain. In the embryo, Fkbp25 gene was strongly expressed in the telencephalon ventricular zone but also in areas active in myogenesis (walls of the ventricle and the atrium) and chondrogenesis (the cartilage of the rib and the hindlimb). An increase in the transcript levels of the Fkbp25 gene was also observed during the two successive proliferation waves of the cerebellum development. Immunostaining on primary cultures of embryonic day 10.5 telencephalon stem cells showed that the Fkbp25 protein was present in the cytoplasm and nuclei of cells cultured for 6h but exclusively in the nuclei of the Tuj-1 immunoreactive neurons obtained after 3 days of culture (The sequence data reported here have been submitted to GenBank under accession no. AF135595.).

Long-sarafotoxins: characterization of a new family of endothelin-like peptides.

Sarafotoxins (SRTXs) constitute a family of vasoactive peptides that were initially isolated from the venom of Atractaspis engaddensis, and that are structurally and functionally related to endothelins (ETs). Analysis of the venom of Atractaspis microlepidota microlepidota revealed several new SRTX molecules manifesting some new structural and functional characteristics. These novel SRTXs are longer by three amino acids than the previously described SRTXs, and are designated here "long-SRTXs". Six isoforms, derived from new poly-cistronic precursors, have been identified so far in the venom of this snake. One of these isoforms, designated SRTX-m, was chemically synthesized and its biological properties were studied. Our results show that SRTX-m induces toxicity in mice, mostly due to vasoconstriction, and also that it has a lower toxicity and potency than the more potent SRTX described up to now: sarafotoxin-b (SRTX-b) from A. engaddensis.

Function-dependent clustering of orthologues and paralogues of cyclophilins.

The 18 kDa archetypal cyclosporin-A binding protein, cyclophilin-A, has multiple paralogues in the human genome. Only 18 of those paralogues have been detected as mRNAs or proteins whose masses vary from 18 to 354 kDa, whereas the functional significance of the open reading frames (ORFs) encoding other paralogues of cyclophilin-A remains unknown. The genomes of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae encode different numbers of the cyclophilin paralogues, some of which are orthologous to the human cyclophilins. A library of novel algorithms was developed and used for computation of the conservation levels for hydrophobicity and bulkiness profiles, and amino acid compositions (AACs) of 303 aligned sequences of cyclophilins. The majority of the paralogues and orthologues encoded in these 6 genomes differ considerably from each other. Some of the orthologues and paralogues have high correlation coefficients (CCFs) for pairwise compared hydrophobicity and bulkiness profiles, and whose AACs differ to a low degree. Convergence of these three properties of the polypeptide chain and apparent conservation of the typical sequence hallmarks and parameters allowed for the clustering of the functionally related orthologues and paralogues of the cyclophilins. The clustering method allowed for sorting out the cyclophilins into several distinct classes. Analyses of the overlapping clusters of sequences permitted delineation of some hypothetical pathways that might have led to the creation of certain paralogues of cyclophilins in the eukaryotic genomes.

Long-size isoelectricfocusing (IEF) in flexible silicone tubes: an application to semi-preparative fractionation of proteins.

Soluble proteins extracted from porcine brains were subjected to a series of optional fractionation steps on various chromatographic media including a novel device for semi-preparative isoelectrofocusing (IEF) carried in a flexible silicone tube. The dimensions of the IEF granulated gel beds can be varied from 40 to 75 cm (length) and 0.4-1.6 cm (diameter) which are dependent on the protein's concentration. An average optimal focalisation time of proteins is dependent of the tube length, its diameter and complexity of proteins' mixtures but it is usually reached during 15,000-30,000 Vh. A series of sequential protein's fractionation techniques including semi-preparative IEF carried in the flexible silicone tube with the following dimensions: 75 cm in length and 1.6 cm in diameter permitted for observation and partial characterisation of several proteins whose expression levels are specifically high in the brain.

A note on clustering the functionally-related paralogues and orthologues of proteins: a case of the FK506-binding proteins (FKBPs).

The expression patterns of 18 FK506-binding proteins (FKBPs) encoded in the human genome have been established whereas the functional significance of the numerous ORFs coding for FKBP-like sequences remains unknown. Nominal masses of the human FKBPs vary from 12 to 135 kDa. Some large FKBPs consist up to four repeats of the 12 kDa FK506-like binding domain (FKBD) whereas other large FKBPs contain one FKBD linked to different functional domains such as TPRs, leucine-zipper, calmodulin-binding domain etc. The genomes of other eukaryotic organisms, namely D. melanogaster, C. elegans, A. thaliana, S. pombe and S. cerevisiae encode different numbers of the FKBPs' paralogues some of which are orthologues to the human FKBPs. A library of novel algorithms was developed and used for computation of the level of conservation of the hydrophobicity and bulkiness profiles, and the amino acid compositions (AACs) of 247 aligned sequences of FKBPs. The pairwisely-compared hydrophobicity and bulkiness profiles for some combinations of the aligned sequences of the FKBDs yielded high values of the correlation coefficients (CCF). The AACs of some combinations of the aligned sequences of the FKBDs also differed to a low degree. The functionally-related orthologues and paralogues of the FKBPs were clustered by using the following criteria: 1 degrees apparent conservation of the crucial amino acid (AA) residues for peptidylprolyl cis/trans isomerase (PPIase) acitity and binding of some immunosuppressive drugs; 2 degrees convergence of the three mentioned above properties of the polypeptide chain; 3 degrees similarity in the sequence attributes pI and total hydrophobicity index (HI). The clustering method was used for setting up several hypotheses on the emergence of certain classes of the FKBPs in the eukaryotic kingdom.

Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity--targets--functions.

Information recovered from genome sequencing projects, multiple sequence alignments, structural analyses of PPIase and published records were used in deciphering the biological diversity, functions and targets of four groups of proteins encoded by dissimilar sets of sequences whose spatial representations exhibit peptidylprolyl cis/trans isomerase activity (PPIase). In the human genome there are encoded fifteen proteins whose segments have significant homology with the sequence of 12 kDa protein which is the target of the potent immunosuppressive macrolides FK506 or rapamycin. The 12 kDa archetype of the FK506-binding protein (FKBP), known as FKBP-12a, is an abundant intracellular protein whereas other FKBPs possessing from one to four FK506-like binding domains (FKBDs) have nominal masses varying from 13 to 135 kDa. The human genome contains at least sixteen genes encoding proteins comprising one cyclosporin-A (CsA) binding domain (CLD) called cyclophilins whose nominal masses vary from 17 to 324 kDa and multiple coding segments for small cyclophilins (17-19 kDa) whose transcription levels and functions remain unknown. The third group of PPIases encoded in the genome comprises two proteins (hPin1 and hParv14) where hPin1 is an important PPIase for cell cycle. The A. thaliana, C. elegans, D. melanogaster and S. cerevisiae genomes encode a less diverse spectrum of PPIases whereas the prokaryotic genomes contain from none to three cyclophilins, from none to four genes encoding FKBPs, one distant homologue of the Pin1 protein named parvulin and the fourth group of PPIases known as trigger factors. PPIases are discretely distributed to different cellular compartments and interact with a number of targets that control a range of cellular processes. Analyses of the sequence alignments of the two groups of PPIases, namely cyclophilins and FKBPs from diverse phyla, show that in each group their sequences diverge but the amino acid residues which form the PPIase activity site and macrolide binding cavity remain well conserved in the majority of them which suggests that the spatial structures and functions of each group of PPIases remain conserved.

Exogenous myristic acid acylates proteins in cultured rat hepatocytes.

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).